Efficacy and safety of certolizumab pegol induction therapy in an unselected Crohn's disease population: results of the FACTS survey.

Background: Switzerland was the first country to approve certolizumab pegol (Cimzia, CZP) for the treatment of patients with moderate to severe Crohn's disease (CD) in September 2007. This phase IV study aimed to evaluate the efficacy and safety of CZP in a Swiss multicenter cohort of practice-based patients. Methods: Baseline and Week 6 evaluation questionnaires were sent to all Swiss gastroenterologists in hospitals and private practices. Disease activity was assessed with the Harvey-Bradshaw Index (HBI) and adverse events were evaluated according to WHO guidelines. Results: Fifty patients (31 women, 19 men) were included; 56% had complicated disease (stricture or fistula) and 52% had undergone prior CD-related surgery. All patients. had prior exposure to systemic steroids, 96% to immunomodulators, 78% to infliximab, and 50% to adalimumab. A significant decrease in HBI was observed at Week 6 (versus Week 0) following induction therapy with CZP 400 mg subcutaneously at Weeks 0, 2, and 4 (12.6 +/- 4.7 Week 0 versus 6.2 +/- 4.4 Week 6, P < 0.001). Response and remission rates at Week 6 were 54% and 40%, respectively. We identified 8/11 CD patients undergoing a 50% fistula response (P = 0.021). The frequency of adverse drug reactions attributed to CZP was 6%. CZP was continued in 80% of patients beyond Week 6. Conclusions: In a population of CD patients with complicated disease behavior, CZP induced a response and remission in 54% and 40% of patients, respectively. This series provides the first evidence of the effectiveness of CZP in perianal fistulizing CD.

Intraperitoneal bevacizumab for control of malignant ascites due to advanced-stage gastrointestinal cancers: A multicentre double-blind, placebo-controlled phase II study - AIO SUP-0108

PURPOSE: Malignant ascites is debilitating for patients with advanced cancer. As shown previously, tumour cell production of vascular endothelial growth factor might be a major cause of the formation of malignant ascites. Intraperitoneal bevacizumab could therefore be an option for symptom control in refractory ascites. PATIENTS AND METHODS: Patients with advanced gastrointestinal cancer and malignant ascites who had undergone paracentesis at least twice within the past 4 weeks were randomly assigned in a 2:1 ratio to intraperitoneal bevacizumab (400 mg absolute) or placebo after paracentesis. During the 8-week treatment period, a minimum interval of 14 d was kept between the applications of the study drug. Primary end-point was paracentesis-free survival (ParFS). RESULTS: Fifty-three patients (median age 63 years) were randomised. Forty-nine patients received at least one study drug application and qualified for the main analysis. The proportion of patients with at least one common toxicity criteria grade III-V event was similar with 20/33 (61%) on bevacizumab and 11/16 (69%) on placebo. Median ParFS was 14 d (95% confidence interval [CI]: 11-17) in the bevacizumab arm and 10.5 d (95% CI: 7-21) on placebo (hazard ratio 0.74, 95% CI: 0.40-1.37; P = 0.16). The longest paracentesis-free period was 19 d on bevacizumab (range 6-66 d) and 17.5 d in the placebo arm (range 4-42) (P = 0.85). Median overall survival was 64 d (95% CI: 45-103) on bevacizumab compared to 31.5 d (95% CI: 20-117) on placebo (P = 0.31). CONCLUSION: Intraperitoneal bevacizumab was well tolerated. Overall, treatment did not result in a significantly better symptom control of malignant ascites. However, patients defined by specific immune characteristics may benefit.

FcγRIIa and FcγRIIIa polymorphisms and cetuximab benefit in the microscopic disease

Purpose: FcγR polymorphisms have been reported to enhance the immune-mediated effects of cetuximab in metastatic colorectal cancer. There are no data on the relationship between these polymorphisms and cetuximab in the early-stage setting. We performed a pharmacogenomic analysis of EXPERT-C, a randomized phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery, and adjuvant CAPOX ± cetuximab in high-risk, locally advanced rectal cancer.

Experimental Design: FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms were analyzed on DNA from peripheral blood samples. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms.

Results: Genotyping was successfully performed in 105 of 164 (64%) patients (CAPOX = 54, CAPOX-C = 51). No deviation from the Hardy–Weinberg equilibrium or association of these polymorphisms with tumor RAS status was observed. FcγRIIa-131R (HR, 0.38; P = 0.058) and FcγRIIIa-158F alleles (HR, 0.21; P = 0.007) predicted improved progression-free survival (PFS) in patients treated with cetuximab. In the CAPOX-C arm, carriers of both 131R and 158F alleles had a statistically significant improvement in PFS (5 years: 78.4%; HR, 0.22; P = 0.002) and overall survival (OS; 5 years: 86.4%; HR, 0.24; P = 0.018) when compared with patients homozygous for 131H and/or 158V (5-year PFS: 35.7%; 5-year OS: 57.1%). An interaction between cetuximab benefit and 131R and 158F alleles was found for PFS (P = 0.017) and remained significant after adjusting for prognostic variables (P = 0.003).

Conclusion: This is the first study investigating FcγRIIa and FcγRIIIa polymorphisms in patients with early-stage colorectal cancer treated with cetuximab. We showed an increased clinical benefit from cetuximab in the presence of 131R and 158F alleles.

TP53 mutational status and cetuximab benefit in rectal cancer: 5-year results of the EXPERT-C trial.

In this updated analysis of the EXPERT-C trial we show that, in magnetic resonance imaging-defined, high-risk, locally advanced rectal cancer, adding cetuximab to a treatment strategy with neoadjuvant CAPOX followed by chemoradiotherapy, surgery, and adjuvant CAPOX is not associated with a statistically significant improvement in progression-free survival (PFS) and overall survival (OS) in both KRAS/BRAF wild-type and unselected patients. In a retrospective biomarker analysis, TP53 was not prognostic but emerged as an independent predictive biomarker for cetuximab benefit. After a median follow-up of 65.0 months, TP53 wild-type patients (n = 69) who received cetuximab had a statistically significant better PFS (89.3% vs 65.0% at 5 years; hazard ratio [HR] = 0.23; 95% confidence interval [CI] = 0.07 to 0.78; two-sided P = .02 by Cox regression) and OS (92.7% vs 67.5% at 5 years; HR = 0.16; 95% CI = 0.04 to 0.70; two-sided P = .02 by Cox regression) than TP53 wild-type patients who were treated in the control arm. An interaction between TP53 status and cetuximab effect was found (P <.05) and remained statistically significant after adjusting for statistically significant prognostic factors and KRAS.

RAS mutations and cetuximab in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: RAS mutations predict resistance to anti-epidermal growthfactor receptor (EGFR) monoclonal antibodies in metastatic colorectal cancer. We analysed RAS mutations in 30 non-metastatic rectal cancer patients treated with or without cetuximab within the 31 EXPERT-C trial.

Methods: Ninety of 149 patients with tumours available for analysis were KRAS/BRAF wild-type, and randomly assigned to capecitabine plus oxaliplatin (CAPOX) followed by chemoradiotherapy, surgery and adjuvant CAPOX or the same regimen plus cetuximab (CAPOX-C). Of these, four had a mutation of NRAS exon 3, and 84 were retrospectively analysed for additional KRAS (exon 4) and NRAS (exons 2/4) mutations by using bi-directional Sanger sequencing. The effect of cetuximab on study end-points in the RAS wild-type population was analysed.

Results: Eleven (13%) of 84 patients initially classified as KRAS/BRAF wild-type were found to have a mutation in KRAS exon 4 (11%) or NRAS exons 2/4 (2%). Overall, 78/149 (52%) assessable patients were RAS wild-type (CAPOX, n = 40; CAPOX-C, n = 38). In this population, after a median follow-up of 63.8 months, in line with the initial analysis, the addition of cetuximab was associated with numerically higher, but not statistically significant, rates of complete response (15.8% versus 7.5%, p = 0.31), 5-year progression-free survival (75.5% versus 67.5%, hazard ratio (HR) 0.61, p = 0.25) and 5-year overall survival (83.8% versus 70%, HR 0.54, p = 0.20).

Conclusions: RAS mutations beyond KRAS exon 2 and 3 were identified in 17% of locally advanced rectal cancer patients. Given the small sample size, no definitive conclusions on the effect of additional RAS mutations on cetuximab treatment in this setting can be drawn and further investigation of RAS in larger studies is warranted.

HER2 in high-risk rectal cancer patients treated in EXPERT-C, a randomized phase II trial of neoadjuvant capecitabine and oxaliplatin (CAPOX) and chemoradiotherapy (CRT) with or without cetuximab.

BACKGROUND: HER2 is an established therapeutic target in breast and gastric cancers. The role of HER2 in rectal cancer is unclear, as conflicting data on the prevalence of HER2 expression in this disease have been reported. We evaluated the prevalence of HER2 and its impact on the outcome of high-risk rectal cancer patients treated with neoadjuvant CAPOX and CRT±cetuximab in the EXPERT-C trial. PATIENTS AND METHODS: Eligible patients with available tumour tissue for HER2 analysis were included. HER2 expression was determined by immunohistochemistry (IHC) in pre-treatment biopsies and/or surgical specimens (score 0-3+). Immunostaining was scored according to the consensus panel recommendations on HER2 scoring for gastric cancer. Tumours with equivocal IHC result (2+) were tested for HER2 amplification by D-ISH. Tumours with IHC 3+ or D-ISH ratio ≥2.0 were classified as HER2+. The impact of HER2 on primary and secondary end points of the study was analysed. RESULTS: Of 164 eligible study patients, 104 (63%) biopsy and 114 (69%) surgical specimens were available for analysis. Only 3 of 104 (2.9%) and 3 of 114 (2.6%) were HER2+, respectively. In 77 patients with paired specimens, concordance for HER2 status was found in 74 (96%). Overall, 141 patients were assessable for HER2 and 6 out of 141 (4.3%) had HER2 overexpression and/or amplification. The median follow-up was 58.6 months. HER2 was not associated with a difference in the outcome for any of the study end points, including in the subset of 90 KRAS/BRAF wild-type patients treated±cetuximab. CONCLUSIONS: Based on the low prevalence of expression as recorded in the EXPERT-C trial, HER2 does not appear to represent a useful therapeutic target in high-risk rectal cancer. However, the role of HER2 as a potential predictive biomarker of resistance to anti-EGFR-based treatments and a therapeutic target in anti-EGFR refractory metastatic colorectal cancer (CRC) warrants further investigation. TRIAL REGISTRATION: ISRCTN Register: 99828560.

Multicenter randomized phase II clinical trial comparing neoadjuvant oxaliplatin, capecitabine, and preoperative radiotherapy with or without cetuximab followed by total mesorectal excision in patients with high-risk rectal cancer (EXPERT-C).

PURPOSE: To evaluate the addition of cetuximab to neoadjuvant chemotherapy before chemoradiotherapy in high-risk rectal cancer. PATIENTS AND METHODS: Patients with operable magnetic resonance imaging-defined high-risk rectal cancer received four cycles of capecitabine/oxaliplatin (CAPOX) followed by capecitabine chemoradiotherapy, surgery, and adjuvant CAPOX (four cycles) or the same regimen plus weekly cetuximab (CAPOX+C). The primary end point was complete response (CR; pathologic CR or, in patients not undergoing surgery, radiologic CR) in patients with KRAS/BRAF wild-type tumors. Secondary end points were radiologic response (RR), progression-free survival (PFS), overall survival (OS), and safety in the wild-type and overall populations and a molecular biomarker analysis. RESULTS: One hundred sixty-five eligible patients were randomly assigned. Ninety (60%) of 149 assessable tumors were KRAS or BRAF wild type (CAPOX, n = 44; CAPOX+C, n = 46), and in these patients, the addition of cetuximab did not improve the primary end point of CR (9% v 11%, respectively; P = 1.0; odds ratio, 1.22) or PFS (hazard ratio [HR], 0.65; P = .363). Cetuximab significantly improved RR (CAPOX v CAPOX+C: after chemotherapy, 51% v 71%, respectively; P = .038; after chemoradiation, 75% v 93%, respectively; P = .028) and OS (HR, 0.27; P = .034). Skin toxicity and diarrhea were more frequent in the CAPOX+C arm. CONCLUSION: Cetuximab led to a significant increase in RR and OS in patients with KRAS/BRAF wild-type rectal cancer, but the primary end point of improved CR was not met.

Two monoclonal antibodies to precisely the same epitope of type ii collagen select non-crossreactive phage clones by phage display: Implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Isolation of HIV-1-neutralizing mucosal monoclonal antibodies from human colostrum.

BACKGROUND: Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. METHODS: We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). RESULTS: The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. CONCLUSIONS: These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.